This invention resides in the field of reference and control materials for hematology instrumentation, with particular attention to simulated human platelets used as controls for automated platelet counting.
Automated blood cell analyzers that provide cell counts for each of the various types of cells present in a sample of blood do so by measurements of the electrical and/or optical properties of each cell type. These properties include electrical impedance, electrical conductance, radio frequency modulation, light scattering, and light absorption, in various combinations. A variety of analyzers are commercially available and used in clinical laboratories, individual analyzers differing in the manner in which they collect and process the data.
Federal regulations require that blood cell analyzers be checked regularly against controls to verify the reliability of the analyzers. The controls are synthetic suspensions that have the certain physical and chemical characteristics similar to those of blood and that include stable cells or particles whose sizes and shapes closely approximate those of the different cells present in human blood. Unfortunately, the different methodologies among the various instruments react to controls in different ways, and certain types of control particles that serve as effective substitutes for one kind of cell on a particular instrument have been found to appear like another type or even like cell debris on a different instrument.
Among the various types of cells in human blood that must be represented in a control, platelets are particularly problematic. The use of actual platelets in the control is unfavorable since platelets disintegrate when the blood in which they are suspended escapes from the vascular system, and the disintegration causes the liberation of thromboplastins that cause blood clotting. In addition, platelets are easily activated and tend to aggregate. They are also expensive. For these reasons, simulated platelets have been developed that are less costly and that lack these unfavorable characteristics. Simulated platelets are typically biological cells or non-biological particles. When biological cells are used, they are cells other than platelets that have been modified to bear characteristics that render them detectable by the same parameters as actual platelets and thereby distinguishable from other cell types. When particles are used, they are particles that have these characteristics. The characteristics differ depending on the methodology of the detection. In some cases, the distinguishing characteristics are size range and size distribution, while in others, the chemical contents, such as a lack of hemoglobin as compared to the presence of hemoglobin in a red blood cell, serve as the differentiating characteristics. For a control to be useful in different types of instruments rather than only one, it is important that the simulated platelet component be detectable as platelets regardless of the methodology of the instrument. This, unfortunately, is not always the case, even when the measured characteristic is size distribution, i.e., it is not uncommon for a particular control to be read as having one size distribution by one means of detection and another by a different means of detection. Fresh human platelets have a log-normal size distribution rather than a Gaussian distribution, and detection instrumentation that relies on particle size also must have either controls that have a log-normal distribution or a computer algorithm that can accept a population that approaches a log-normal size distribution.
A relatively inexpensive substitute for actual human platelets and one that lacks the disintegration and aggregation characteristics of human platelets are red blood cells from non-human vertebrates. To render these cells useful as simulated platelets, the cells are reduced in size and treated with a fixing agent to toughen the cell membranes. Goats are a favored source of red blood cells as substitutes for platelets, since goat red blood cells can either be altered or blended to a size and size distribution similar to those of human platelets. One method of size adjustment is the suspension of the cells in a hyperosmotic solution to draw cellular fluid from the cells by osmotic pressure. The fixing treatment is performed either before or after the use of osmotic pressure, depending on whether the fixing treatment is used as a means of controlling or limiting the rate of passage of cellular fluid when osmotic pressure is applied.
When the fixing treatment is done to control the rate of fluid passage through the cell membrane, the purpose is to achieve a desired particle size range and size distribution. Achieving a particular particle size range and distribution as measured by optical techniques however does not always result in the same size range and distribution as measured by electrical techniques. One method of correcting this deficiency is disclosed by Ryan, W. L. (Streck Laboratories, Inc.), U.S. Pat. No. 5,008,201, issued Apr. 16, 2001. This method involves preparing a graduated series of particle sizes by subjecting different populations of cells to different degrees of fixation to cause each population to shrink to a different degree when osmotic pressure is applied. The populations are then combined in proportions that will collectively approximate the desired size distribution. This is labor-intensive and susceptible to flag in both the selected proportions and the differing degrees of treatment.
It has been discovered that platelet analogs with a size range and distribution detected by optical measurement that conform closely to the size range and distribution detected by electrical measurement can be prepared from red blood cells of a non-human vertebrate animal that have already been shrunken in size and then fixed. The preparation method involves first heating the size-reduced and fixed cells to a temperature above ambient temperature for a period of time sufficient to have a denaturing effect on the cells, then exposing the cells to a fixing agent, preferably before or after cooling the cells to a temperature at or close to ambient temperature. Effective results with this method can be achieved without the need for separately preparing distinct lots of cells of different sizes and combining the lots in carefully selected proportions to achieve a combination that approximates the desired size distribution. The process can instead be performed on a single lot and still achieve the desired distribution as detected by both electrical and optical modes of measurement. This preparation method brings the size and distribution characteristics closer to those that enable the shrunken cells to be used as controls for evaluating actual human platelets.
U.S. Pat. No. 4,179,398 and U.S. Patent Application Publication No. 2006/0223187 describe methods for preparing human platelet analogs using goat red blood cells as starting material. The present invention provides a new process for producing simulated human platelets of more desirable characteristics, such as improved optical properties and a more consistent reading in size range and distribution between optical and electrical measurements.